解析CKLF1引起颈动脉生物钟节律凌乱诱发血管内膜增生的分子机制

It is estimated that 70%-80% of the stroke patients are caused by carotid atherosclerotic plaque in China. Previously, we had found that chemokine like factor 1 (Cklf1) is highly expressed in carotid plaques of human and increased CKLF1 secretion is associated with vascular restenosis in rat carotid artery injury model. Cklf1 plays an important role in promoting migration and proliferation of VSMC. CKLF1 activates the phosphatidylinositol signaling pathway and upregulates the expression and activation of AKT1, a specific initiator of circudian clock in blood vessels. Noteworthy, phospho-AKT1 stimulated the nuclear exportation of BMAL1 that is a critical transcription factor in circadian clock. BMAL1 KO rat showed a remarkably increased intimal hyperplasia accompanied with the dysfunction of vascular circadian clocks. Thus, we provided a hypothesis that abnormal expression of CKLF1 disrupted rhythmic chromatin modification regulated by BMAL1, resulting in a large number of gene expression related to cell proliferation and migration and consequently atherosclerosis and restenosis. The aim of this project is to investigate the effect of CKLF1 on circadian rhythms and to analyze the role of circadian rhythmic disorder on VSMC proliferation using smooth muscle cell specific Cklf1 knockout mice after injury. We believe that this study might take insight into a new theory for prevention of atherosclerosis and restenosis in the future, and further is beneficial to lay a theoretical foundation for the development of biological clock rhythm related drugs to treat both diseases.

流行病学研究表明，多达70-80%的脑卒中患者是由颈动脉粥样硬化斑块造成的血管狭窄所诱发。前期研究发现，趋化素样因子1（Cklf1）在颈动脉斑块中高量表达，大鼠颈动脉损伤处分泌CKLF1增加促进了血管内膜增生。CKLF1激活了血管平滑肌中磷脂酰肌醇信号通路，上调了血管生物钟发生器AKT1的表达和激活。p-AKT1促使生物钟核心转录因子BMAL1出核，破坏受时因子的表达节律，而小鼠血管生物钟凌乱可导致内膜增生，诱发动脉硬化。由此，我们推测CKLF1引发BMAL1调控的节律性染色质修饰变化出现异常，最终造成动脉粥样硬化和术后再狭窄。为验证此假设，本项目拟利用平滑肌细胞特异Cklf1敲除小鼠颈动脉损伤模型，通过在体、离体实验探讨CKLF1对血管生物钟的作用并解析生物钟节律凌乱对血管平滑肌细胞增殖的调控机制。为血管内膜增生性疾病的防治提供了新的理论基础并为今后生物钟药物增加新的适应症。

所有感光的生物体，不论是简单到蓝藻还是复杂如人类，都存在各自的“生物计时器”-生物钟。生物钟调控着生命体的生物学行为，如睡眠觉醒节律、饥饿进食节律等。近年来的研究发现，生物钟基因可作为转录因子调节许多基因的表达，从而参与多种生理过程。外界刺激及不良生活方式会导致正常生物节律的紊乱，进而导致各种疾病的发生。.既往研究中我们揭示了CKLF1分子通过激活PI3K/AKT/NF-B通路，促进平滑肌增殖从而导致内膜增生。近年来，国际上有研究报道AKT分子是血管系统内的节律发生器；也有许多研究证实生物节律的紊乱（如Bmal或Clock基因功能缺陷）会导致平滑肌生理功能的改变，从而导致动脉粥样硬化、动脉瘤等病变。然而，CKLF1作为炎症因子，其对于血管生物节律的影响尚不清楚。在本研究中我们证实，CKLF1分子激活AKT，活化的AKT分子磷酸化BMAL1分子，从而导致BMAL1分子的出核。BMAL1分子作为转录因子，出核导致其失去转录活性，导致下游调控的前列腺素合酶2基因表达降低，从而导致平滑肌增殖，促进内膜增生的发生。